The ASH1 HOMOLOG 2 (ASHH2) histone H3 methyltransferase is required for ovule and anther development in Arabidopsis.

BACKGROUND: SET-domain proteins are histone lysine (K) methyltransferases (HMTase) implicated in defining transcriptionally permissive or repressive chromatin. The Arabidopsis ASH1 HOMOLOG 2 (ASHH2) protein (also called SDG8, EFS and CCR1) has been suggested to methylate H3K4 and/or H3K36 and is similar to Drosophila ASH1, a positive maintainer of gene expression, and yeast Set2, a H3K36 HMTase. Mutation of the ASHH2 gene has pleiotropic developmental effects. Here we focus on the role of ASHH2 in plant reproduction. METHODOLOGY/PRINCIPAL FINDINGS: A slightly reduced transmission of the ashh2 allele in reciprocal crosses implied involvement in gametogenesis or gamete function. However, the main requirement of ASHH2 is sporophytic. On the female side, close to 80% of mature ovules lack embryo sac. On the male side, anthers frequently develop without pollen sacs or with specific defects in the tapetum layer, resulting in reduction in the number of functional pollen per anther by up to approximately 90%. In consistence with the phenotypic findings, an ASHH2 promoter-reporter gene was expressed at the site of megaspore mother cell formation as well as tapetum layers and pollen. ashh2 mutations also result in homeotic changes in floral organ identity. Transcriptional profiling identified more than 300 up-regulated and 600 down-regulated genes in ashh2 mutant inflorescences, whereof the latter included genes involved in determination of floral organ identity, embryo sac and anther/pollen development. This was confirmed by real-time PCR. In the chromatin of such genes (AP1, AtDMC1 and MYB99) we observed a reduction of H3K36 trimethylation (me3), but not H3K4me3 or H3K36me2. CONCLUSIONS/SIGNIFICANCE: The severe distortion of reproductive organ development in ashh2 mutants, argues that ASHH2 is required for the correct expression of genes essential to reproductive development. The reduction in the ashh2 mutant of H3K36me3 on down-regulated genes relevant to the observed defects, implicates ASHH2 in regulation of gene expression via H3K36 trimethylation in chromatin of Arabidopsis inflorescences.

T-loop phosphorylation of Arabidopsis CDKA;1 is required for its function and can be partially substituted by an aspartate residue.

As in other eukaryotes, progression through the cell cycle in plants is governed by cyclin-dependent kinases. Phosphorylation of a canonical Thr residue in the T-loop of the kinases is required for high enzyme activity in animals and yeast. We show that the Arabidopsis thaliana Cdc2(+)/Cdc28 homolog CDKA;1 is also phosphorylated in the T-loop and that phosphorylation at the conserved Thr-161 residue is essential for its function. A phospho-mimicry T161D substitution restored the primary defect of cdka;1 mutants, and although the T161D substitution displayed a dramatically reduced kinase activity with a compromised ability to bind substrates, homozygous mutant plants were recovered. The rescue by the T161D substitution, however, was not complete, and the resulting plants displayed various developmental abnormalities. For instance, even though flowers were formed, these plants were completely sterile as a result of a failure of the meiotic program, indicating that different requirements for CDKA;1 function are needed during plant development.

A positive signal from the fertilization of the egg cell sets off endosperm proliferation in angiosperm embryogenesis.

Double fertilization of the egg cell and the central cell by one sperm cell each produces the diploid embryo and the typically triploid endosperm and is one of the defining characteristics of flowering plants (angiosperms). Endosperm and embryo develop in parallel to form the mature seed, but little is known about the coordination between these two organisms. We characterized a mutation of the Arabidopsis thaliana Cdc2 homolog CDC2A (also called CDKA;1), which has a paternal effect. In cdc2a mutant pollen, only one sperm cell, instead of two, is produced. Mutant pollen is viable but can fertilize only one cell in the embryo sac, allowing for a genetic dissection of the double fertilization process. We observed exclusive fertilization of the egg cell by cdc2a sperm cells. Moreover, we found that unfertilized endosperm developed, suggesting that a previously unrecognized positive signal from the fertilization of the egg cell initiates proliferation of the central cell.